Obesity, or an excess of body fat relative to lean body mass, is a serious health problem in the United States and abroad. A person is clinically obese if he or she has excess adipose tissue. More particularly, for purposes of the present invention, a person is obese if the person's body mass index equals or exceeds 27 kg/m2 and the person has excess adipose tissue.
Statistics suggest that more than 25% of the United States population and 27% of the Canadian population are overweight. Complications of obesity include, among others, diabetes mellitus, hypertension, hyperlipoproteinemia, cardiac diseases (atherosclerotic disease, congestive heart failure, etc.), pulmonary diseases (e.g., sleep apnea, restrictive lung disease), cerebrovascular injury, cancers (including breast, uterine, colon, and prostate), gall bladder disease (stones, infection), toxemia during pregnancy, risks during surgery (e.g., pneumonia, wound infection, thrombo-phlebitis), gout, decreased fertility, degenerative arthritis, and early mortality. Psychological complications of obesity include poor self-image and poor body-image. Social complications of obesity include discrimination in jobs, education and marriage. Despite the known associated risks, a significant portion of the population is unable to lose weight or maintain weight loss. Obesity is now considered the second leading cause of preventable death in the United States, second only to smoking, with an estimated 300,000 deaths annually. Accordingly, reduction of the prevalence of obesity in the adult population to less than 20% is included by the US Department of Health and Human Services among the national health objectives.
The human tragedy notwithstanding, the monetary costs of obesity are staggering. The total cost attributable to obesity in 1995 has been estimated to be in excess of $99 billion, with approximately $51.64 billion paid in direct medical costs. Overall, the direct costs associated with obesity represent 5.7% of the annual United States national health expenditure. Thus, it is clear that the magnitude of this problem produces a significant demand for safe and effective treatments for obesity. Obesity has a number of known and suspected etiologies. See A. Sclafani, “Animal Models of Obesity: Classification and Characterization,” Int. J. Obesity 8, 491-508 (1984); G. A. Bray, “Classification and Evaluation of the Obesities,” Med. Clin. N. Am. 73, 161-184 (1989). While it is generally known that overeating and inactivity are factors that lead to obesity, there is substantial evidence of genetic contribution to obesity. Although the molecular characterization of genetic pathways associated with obesity is incomplete, several recent advances into the elucidation of these pathways have been made. Research indicates that there are several genes that act independently or in combination to modulate metabolic pathways associated with excess adipose tissue accumulation. The presence of these various pathways suggests a complex system of obesity regulation, a system that has not yet been fully defined.
Some mouse models for obesity include obese (ob/ob), agouti (Ay/a), tubby (tub), fat (fa/fa) and diabetes (db/db). These models have proven to be effective in the molecular characterization of these genetic loci because of their ability to simplify the heritability of complex traits.
One gene responsible for the autosomal recessive mouse obesity mutation tub has been identified by positional cloning and shown to be associated with maturity-onset obesity (U.S. Pat. No. 5,776,762). Identification of the tub gene and the protein it encodes may lead to the development of agents that will function to modulate either the protein or gene expression. However, a disadvantage of this system is the ubiquitous nature of the gene, in that the gene is expressed in high levels in the brain, eye and testis, and at lower levels in various adult and fetal tissues, including small and large intestine, ovary and adipose tissue. Although the gene may be used as a probe for identification of other tubby polypeptides, development of agents to modulate the expression of these polypeptides would not be specific to a particular tissue.
Similarly, the ob gene has recently been cloned. The ob gene encodes a protein known as leptin, which has been implicated in an energy feedback loop responsible for controlling vertebrate energy balance. Serum levels of leptin are increased proportionately to excess adipose accumulation as a result of increased expression in hypertrophic fat cells in obese patients. In vitro studies have indicated that insulin and glucocorticoids upregulate leptin mRNA expression in a synergistic manner. The subsequent expression of the protein product thereby functions to stimulate metabolic activity. The promoter of the ob gene has been cloned and is a candidate for pharmacological control (U.S. Pat. No. 6,124,448).
In addition to cloning the promoter of the ob gene, attempts at obesity regulation have also been made through modulation of the ob gene. The ob/ob mouse is a model of obesity and diabetes that carries an autosomal recessive trait linked to a mutation in the sixth chromosome (Yiying Zhang et al. Nature 372: 425-32 (1994). Pharmacological agents have therefore been developed to mimic the action of the ob gene encoded protein and assist in regulation of appetite and metabolism. However, the majority of obese humans actually have normal or somewhat elevated levels of leptin as compared to lean humans leading some to hypothesize that human obesity may be more related to leptin resistance rather than leptin deficiency. Recent clinical trials have shown that leptin may be useful for a certain subset of patients, but not for the treatment of obesity generally (Gura, T., Science 1999, 286 (5441): 881-2).
Using molecular and classical genetic markers, the ob and db genes have been mapped to proximal chromosome 6 and midchromosome 4, respectively (Bahary et al., Proc. Nat. Acad. Sci. USA, 87:8642-8646 (1990); Friedman et al., Genomics, 11:1054-1062 (1991)). In both cases, the mutations map to regions of the mouse genome that are syntenic with human, which suggested that if there were human homologs of ob and db, they would likely map, respectively, to human chromosomes 7q and 1p. In fact, the human homologs have been positionally cloned—OB (the human homolog for ob) has been cloned to human chromosome 7q31.3 (Isse, et al. J Biol Chem 1995 Nov. 17; 270 (46): 27728-33). LEPR (the human homolog for db) has been mapped to human chromosome 1p31 (Thompson, et al. Genomics 1997; 39(2):227-30). Defects in the leptin receptor gene results in obesity in other mammalian species: the fa gene in the rat encodes the leptin receptor.
Further, a method for detecting differential expression of specific gene loci has been suggested as a method for identifying a compound that modulates gene expression, but specific proteins and pathways have not yet been identified.
Traditionally, pharmacological approaches to weight loss or prevention of weight gain have relied either on reduction of food intake or on reduction of nutrient absorption. Drugs of the first group, which include Redux (American Home Products) and Meridia (Knoll Pharmaceuticals), affect neurotransmitter activity in the brain, resulting in appetite suppression and decreased food intake. While effective in producing a moderate weight loss in some proportion of patients these medications are associated with a number of adverse side effects.
Drugs of the second group, including Xenical (Hoffmann-La Roche), reduce total absorption of fat from the gastrointestinal tract. However, inhibition of fat absorption by this drug can lead to avitaminosis since successful uptake of fat soluble vitamins from the intestines is impaired in the absence of fat. Additionally, these drugs produce unpleasant side-effects, such as steatorrhea, which reduce patient compliance. Other health problems have been shown to stem directly or indirectly from use of the drug as well such as an increased incidence of breast cancer.
Consequently, focus has since shifted away from these group one and two pharmaceuticals and instead towards targeting suppression of gene expression or protein inhibition. Some pharmaceutical examples are leptin (Amgen), leptin receptor (Progenitor) and tubby (Millennium Pharmaceuticals). However, expression of these genes is not limited to adipose tissue and many specifically act on the brain to stimulate or decrease adipose accumulation. Therefore, it is possible that development of drugs to specifically target the central nervous system (CNS) to interfere with the CNS-active pathways in obese patients may produce similar side effects to those appetite suppressors that are currently available. Thus, it is extremely beneficial to target adipose specific genes or the proteins encoded by such genes. Moreover, treating obese or overweight subjects with compounds that target genes or the proteins they encode that regulate the ability of the adipocyte to store fat will result in a decrease in adipose mass and a positive impact on the subject's health. The natriuretic peptide clearance receptor represents one such gene/protein.
The natriuretic peptide (NP) system is an important component in the regulation of sodium and water balance, blood volume and blood pressure. This system works through several mechanisms, for example decreasing renin release and consequently aldosterone release by the adrenal cortex, thereby decreasing sodium and fluid retention in the kidneys. The NP system has also been shown to inhibit vasopressin, again causing a decrease in fluid retention in the kidney. These actions contribute to reductions in blood volume, and therefore central venous pressure, cardiac output, and arterial blood pressure. A third mechanism appears to be arterial vasodilation in response to hypervolemia.
The family of natriuretic peptides includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The primary signaling molecules for these peptides are natriuretic peptide receptor A (nprA, npr1) which binds ANP and BNP, and natriuretic peptide receptor B (nprB, npr2) which binds CNP. The A and B receptors share approximately 62% identity at the amino acid level and have been classified as guanylyl cyclase receptors. That is, their intracellular domains possess a kinase-like domain and a guanylyl cyclase catalytic domain. Upon ligand binding to the extracellular ligand binding domain, the guanylyl cyclase catalytic domain is activated causing and increase in intracellular cyclic GMP (cGMP) which potentiates the physiological activity of the receptors. The A receptor has been implicated in vasodilation, increased diuresis and natriuresis, and decreased renin and aldosterone. The B receptor has been implicated in vasodilation and increased long bone growth. (for review see Levin, et al., New England Journal of Medicine 339(5): 321-328 (1998) and Potter, et al., J. Biol. Chem. 276(9): 6057-6060 (2001)).
There is a third receptor, natriuretic peptide receptor C, (nprC, npr3) which shares only approximately 32% identity with nprA and nprB over the length of the protein. The C receptor retains a similar extracellular ligand binding domain and trans-membrane domain, but has only a short intracellular domain (37 amino acid residues) which lacks both the kinase-like domain and guanylyl cyclase activation domain. ANP, BNP, and CNP all bind this receptor with approximately equal affinity. The receptor is called the natriuretic peptide clearance receptor in that it has been shown to participate in the local clearance of the natriuretic peptides. The receptor binds the ligand and is internalized. The ligand is degraded and the receptor is retroendocytozed back to the cell surface (Nussenzveig, et al., J. Biol. Chem. 265(34) 20952-20958 (1990). The C receptor accounts for approximately 50% of natriuretic peptide clearance, the other half being carried out by cell surface neutral endopeptidases. However, while nprA and nprB are often called biologically active receptors to the exclusion of nprC, it has been suggested that nprC has other biological activity other than simply natriuretic peptide clearance. Several groups have shown that the c-terminal domain of nprC can interact with inhibitory G-proteins (Gi) that act to downregulate adenylyl cyclase and thus reduce the level of intracellular cyclic A MP (cAMP) (Palaparti, et al., Biochem. J., 346:313-320 (2000) and Pagano, et al., J. Biol. Chem. 276(25):22064-22070 (2001)). Recently, it has been postulated that the vasodilatory effects of endothelium-derived hyperpolarizing factor (EDHF) may be attributed to such nprC mediated adenylyl cyclase inhibition (Chauhan, et al., Proc. Natl. Acad. Sci. USA, 100(3):1426-1431 (2003).
ANP and BNP have been linked to lipolysis in a cGMP dependent manner which does not depend on cAMP production or phosphodiesterase inhibition (Sengenes, et al., FASEB J., 14(10):1345-51 (2000)). Thus, ANP and BNP, but not nprC, have been implicated in the biology of the adipocyte. In this case, for example, the authors have attributed the lipolytic effects as being linked to cGMP production in which nprC does not participate.
Specific npr3 knockout mice were made to determine the effect of an absence of nprC on water balance, salt balance, and blood pressure (Matsukawa, et al., Proc. Natl. Acad. Sci. USA 96:7403-7408 (1999)). The animals have a moderately but statistically significantly lowered blood pressure and with age show an increase in daily water uptake with a significant increase in urinary output. The knockout mice also have a defect in the ability to concentrate their urine. The observed alterations in renal function were interpreted as being the result of a failure of local clearance of natriuretic peptides in the glomerular and post-glomerular structures resulting in an increase in filtered volume and a decrease in water reabsorption. The decrease in blood pressure was attributed to simple hypovolemia. These animals exhibit skeletal abnormalities including an overgrowth of the long bones as well as other defects. The authors note that the animals exhibit “elongated femurs, tibias, metatarsal, and digital bones, longer vertebral bodies, increased body length, and decreased weight [emphasis added].” However, the authors did not account for the decrease in weight nor did they make any examination of the adipose tissue.
Several spontaneously occurring mutants in the npr3 have been identified, the first of which was called longjohn (lgj) due to the skeletal defects described above. A French group studied them to examine and compare the skeletal defects among the three strains (Jaubert, et al., Proc. Natl. Acad. Sci. USA 96:10278-10283 (1999). The authors note offhandedly that “ . . . older mutant mice are exceptionally thin and at necropsy normal body fat deposits are absent.” Again, the authors did not make any more mention of the animals' weight or adipose tissue.